WO2017009837A1 - Micro-arn pour le traitement des tumeurs solides malignes et des métastases - Google Patents

Micro-arn pour le traitement des tumeurs solides malignes et des métastases Download PDF

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WO2017009837A1
WO2017009837A1 PCT/IL2016/050759 IL2016050759W WO2017009837A1 WO 2017009837 A1 WO2017009837 A1 WO 2017009837A1 IL 2016050759 W IL2016050759 W IL 2016050759W WO 2017009837 A1 WO2017009837 A1 WO 2017009837A1
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mir
cancer
pharmaceutical composition
mirna
administering
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PCT/IL2016/050759
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Avital GILAM
Noam Shomron
Eitan Friedman
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Ramot At Tel Aviv University Ltd.
Tel Hashomer Medical Research Infrastructure And Services Ltd.
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Priority to EP16823987.9A priority Critical patent/EP3322423A4/fr
Priority to US15/744,086 priority patent/US10568901B2/en
Publication of WO2017009837A1 publication Critical patent/WO2017009837A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention relates to a micro-RNA (miRNA)-based treatment of cancer, particularly solid tumors, and metastasis.
  • miRNA micro-RNA
  • Malignant solid tumors are masses of abnormal tissue that originate in organs or soft tissues that typically do not include fluid areas and cysts. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Malignant solid tumors initially grow in the organ of their cellular origin. However, such cancers may spread to other organs through metastatic tumor growth in advanced stages of the disease.
  • miRNAs tissue specific micro-RNAs
  • miRNAs are non-coding RNA molecules of about 21-22 nucleotides in length, which are endogenously expressed either ubiquitously or in a tissue-specific manner and play an important regulatory role in various cellular processes.
  • miRNAs regulate gene expression by binding to complementary sequences in target messenger RNA (mRNA) molecules, typically in untranslated regions (UTRs) thereof, and triggering their repression.
  • mRNA target messenger RNA
  • UTRs untranslated regions
  • miR-96 and miR-182 are two miRNAs which are part of a cluster of miRNAs containing miRs-183, 96 and 182.
  • the miR-183/-96/-182 cluster is a highly conserved polycistronic miRNA cluster shown to be abnormally expressed in a variety of tumors.
  • US 2014/0088170 discloses differentially expressed microRNA molecules for the treatment and diagnosis of cancer, particularly identification of several miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. Among others, differential expression of miR-96 and miR-182 in cancer cells compared to normal cells is disclosed.
  • the present invention provides compositions and methods for effective treatment of malignant solid tumors in a subject, to reduce or even prevent the formation of metastasis, using micro-RNAs, namely miR-96 and/or miR-182.
  • the present invention is based in part on the finding that over-expression of miR- 96 or miR-182 in cancer cells in-vitro resulted in reduced migration and invasion of the cells through a membrane, as compared to over-expression of a control RNA sequence.
  • Over-expression of miR-96 and/or miR-182 in breast cancer cells in an in-vivo model of breast cancer metastasis almost completely abolished metastasis formation compared to a control RNA sequence.
  • over-expression of just one of the two miRs was sufficient to exert the beneficial anti-metastatic effect. Further improvement may be achieved by using both miR-96 and miR-182.
  • the present invention discloses for the first time that successful treatment with miR-96 and/or miR-82 depends on the genotype at a specific polymorphic position within a target gene of these miRs, denoted PALLD.
  • the PALLD gene (PALLD Gene ID: 23022, accession No: NG_013376), encoding for palladin protein (Accession No: Q8WX93), which is involved in cytoskeleton rearrangement, has a G/C single nucleotide polymorphism (SNP) within the binding site of miR-96 and miR-182, identified by reference number rsl071738 (dbSNP database). It is now disclosed that the C allele is required for efficient regulation of this gene by the miRs. Without being bound by any particular theory or a mechanism of action, it is contemplated that the miRNA molecules down- regulate palladin expression, and thus inhibit and even completely abolish the ability of the primary tumor to metastasize.
  • SNP G/C single nucleotide poly
  • the present invention provides a method for reducing or preventing cancer metastasis in a subject in need thereof, the method comprising administering to the subject at least one miRNA molecule selected from the group consisting of miR-96 and miR-182, or at least one vector expressing or encoding the same, thereby reducing or preventing cancer metastasis in the subject.
  • the subject according to the present invention is typically a human subject diagnosed with cancer.
  • the subject is preferably an individual with the C allele of the polymorphic site within the PALLD gene identified by reference number rsl071738.
  • the method further comprises determining that the subject is carrying the C allele of the single nucleotide polymorphism (SNP) rsl071738 prior to administering the at least one miRNA molecule or the at least one vector expressing or encoding the same.
  • SNP single nucleotide polymorphism
  • the subject is at risk of developing metastasis and the administering is carried out prior to metastasis formation. In other embodiments, the subject has already developed metastases and the administering is carried out after metastasis formation.
  • the method comprises administering a miR-96 molecule and/or a miR-182 molecule, or vectors expressing or encoding the miR-96 and/or miR-182 molecules.
  • the miR-96 molecule and the miR-182 molecule, or the vectors expressing or encoding the same are administered concomitantly.
  • the miR-96 molecule and the miR- 182 molecule, or the vectors expressing or encoding the same are administered sequentially.
  • the miR-96 and the miR-182 are expressed or encoded by a single vector.
  • the miR-96 is a mature miR-96 as set forth in SEQ ID NO:l (5 '-UUUGGCACUAGC ACAUUUUUGCU) .
  • the miR-96 is a precursor of miR-96.
  • the precursor of miR-96 is a pri-miRNA as set forth in SEQ ID NO: 2.
  • the precursor of miR-96 is a pre-miRNA as set forth in SEQ ID NO: 3.
  • the miR-182 is a mature miR-182 as set forth in SEQ ID NO: 4 (5 - UUUGGCAAUGGUAGAACUCACACU).
  • the miR-182 is a precursor of miR-182.
  • the precursor of miR-182 is a pri-miRNA as set forth in SEQ ID NO: 5.
  • the precursor of miR-182 is a pre-miRNA as set forth in
  • the at least one miRNA molecule or vector expressing or encoding the same is formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier.
  • the administering is administering systemically. In other embodiments, the administering is administering locally. In some embodiments, administering locally is administering into a tumor. In additional embodiments, administering locally is administering into a space or cavity adjacent to a tumor. In other embodiments, administering locally is administering into a space or cavity formed after tumor resection.
  • the present invention provides a pharmaceutical composition comprising at least one miRNA molecule selected from the group consisting of miR-96 and miR-182, or at least one vector expressing or encoding the same, for use in reducing or preventing breast cancer metastasis.
  • the pharmaceutical composition is formulated for systemic administration. In other embodiments, the pharmaceutical composition is formulated for local administration. In some embodiments, the pharmaceutical composition is formulated for intra-tumor administration.
  • the present invention provides a miRNA molecule selected from the group consisting of miR-96 and miR-182, or a vector expressing or encoding the same, for use in reducing or preventing cancer metastasis.
  • FIGS. 2A-D Regulation of palladin by miR-182 (Figs. 2A-B) and miR-96 (Figs. 2C-D) in a Renilla/ Firefly Luciferase reporter assay.
  • FIGS. 3A-H Regulation of endogenous palladin levels in Hs578 cells (Figs. 3A- E) and 4T1 cells (Figs. 3F-H) over-expressing miR-96, miR-182 or a scrambled control sequence.
  • FIGS 4A-D Migration and invasion of cells over-expressing miR-96, miR-182 or a scrambled control sequence.
  • FIG. 4A Wound healing assay, Hs578 cells. Left panel - pictures (five fields) taken at the indicated time points following scratch. Right panel - percentage of open wound at each time point compared to time 0;
  • FIG. 4B Matrigel invasion assay, Hs578 cells. Left panel - representative fields. Right panel - invasion rate relative to control;
  • FIG. 4C Transwell migration assay, 4T1 cells. Left panel - representative fields. Right panel - migration rate relative to control;
  • FIG. 4D Matrigel invasion assay, 4T1 cells. Left panel - representative fields. Right panel - invasion rate relative to control.
  • Figures 5A-F Effect of down-regulation of miR-96 or miR-182 on the mRNA levels of palladin (Figs. 5A-C) and cell migration (Fig. 5D) in MCF7 cells; and on cell migration (Fig. 5E) and invasion (Fig. 5F) in 4T1 cells. Ctrl- scrambled control sequences; Standard errors are shown in Figs. 5A-C.
  • FIG. 6A Palladin mRNA expression level following stable over-expression of palladin shRNA (Palladin KD) or a scrambled shRNA (Ctrl) in 4T1 cells
  • FIG. 6B Palladin protein level following stable over-expression of palladin shRNA (Palladin KD) or a scrambled shRNA (Ctrl) in 4T1 cells
  • FIG. 6C Transwell migration assay
  • FIG. 7A-C Effect of over-expression of miR-96/miR-182 or anti-miR-96/anti- miR-182 on cell proliferation rate in 4T1 cells (Fig. 7A); MCF-7 cells (Fig. 7B); and Hs578 cells (Fig. 7C), transfected with the indicated miR. Paired student t-test was used for statistical analysis.
  • FIGS. 8A-D MiR levels (Figs. 8A-B) and palladin expression (Figs. 8C-D) in primary tumors from mice injected with 4T1 cells stably transformed with miR-96, miR- 182 or a scrambled RNA molecule.
  • Figures 9A-F Parameters of primary tumors in mice injected with 4T1 cells stably transformed with miR-96, miR-182 or a scrambled RNA molecule.
  • Figs. 9A-C Tumor volume, tumor diameter and mice weight starting from day 5 following inoculation of the tumor cells;
  • Fig. 9D exemplary fluorescence measurements at the removal day of the tumors;
  • Figs. 9E-F average tumor area and average weight at the day of their removal.
  • FIGS 10A-E Lung metastasis in mice injected with 4T1 cells stably transformed with miR-96, miR-182 or a scrambled RNA molecule.
  • FIG. 10A Exemplary CT photos of lungs. Metastatic nodules are marked with an arrow;
  • Figs. lOB-C Average quantity of lung metastatic nodules and average diameter of lung metastases;
  • Figs. 10D- E Exemplary fluorescence measurements in lungs and the average percentage of fluorescent area per lung. Metastatic areas are circled with white line.
  • compositions and methods for treating cancer in a subject More particularly, the compositions and methods of the present invention are particularly useful for inhibiting and even preventing cancer metastasis.
  • the compositions and methods according to embodiments of the present invention utilize specific miRNAs, namely, human miR-96 (also termed hsa- miR-96) and/or human miR-182 (also termed hsa-miR-182).
  • miRNAs namely, human miR-96 (also termed hsa- miR-96) and/or human miR-182 (also termed hsa-miR-182).
  • Such compositions and methods are particularly useful for treating cancer and cancer metastasis, as exemplified herein.
  • nucleic acid As referred to herein, the terms “nucleic acid”, “nucleic acid molecules” “oligonucleotide”, “polynucleotide”, and “nucleotide” may interchangeably be used herein.
  • the terms are directed to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded, double stranded, triple stranded, or hybrids thereof.
  • the term also encompasses RNA/DNA hybrids.
  • the polynucleotides may include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA.
  • the DNA or RNA molecules may be, for example, but not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, Antisense RNA, and the like. Each possibility is a separate embodiment.
  • the terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent internucleoside linkages, as well as oligonucleotides having non-naturally occurring portions, which function similarly to respective naturally occurring portions.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • construct refers to an artificially assembled or isolated nucleic acid molecule which may include one or more nucleic acid sequences, wherein the nucleic acid sequences may include coding sequences (that is, sequence which encodes an end product), regulatory sequences, non-coding sequences, or any combination thereof.
  • construct includes, for example, vector but should not be seen as being limited thereto.
  • vector refers to recombinant constructs engineered to encode or express polynucleotides in a target cells, such as DNA, RNA, miRNA, shRNA, siRNA, antisense oligonucleotides, and the like.
  • Vectors may include such vectors as, but not limited to, viral and non-viral vectors, plasmids, and the like.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being expressed in a host cell.
  • Expression vectors typically contain a variety of "control sequences,” which refer to nucleic acid sequences necessary, for example, for the transcription of an operably linked coding or non-coding sequence in a particular host organism.
  • control sequences refer to nucleic acid sequences necessary, for example, for the transcription of an operably linked coding or non-coding sequence in a particular host organism.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • an expression vector can be used to encode for or express one or more miRNA molecules in a target cell.
  • the term "complementarity" is directed to base pairing between strands of nucleic acids.
  • each strand of a nucleic acid may be complementary to another strand in that the base pairs between the strands are non- covalently connected via two or three hydrogen bonds.
  • Two nucleotides on opposite complementary nucleic acid strands that are connected by hydrogen bonds are called a base pair.
  • adenine (A) forms a base pair with thymine (T) and guanine (G) with cytosine (C).
  • thymine is replaced by uracil (U).
  • the degree of complementarity between two strands of nucleic acid may vary, according to the number (or percentage) of nucleotides that form base pairs between the strands. For example, “100% complementarity” indicates that all the nucleotides in each strand form base pairs with the complement strand. For example, “95% complementarity” indicates that 95% of the nucleotides in each strand from base pair with the complement strand.
  • the term sufficient complementarity may include any percentage of complementarity from about 30% to about 100%.
  • introducing and “transfection” may interchangeably be used and refer to the transfer of molecules, such as, for example, nucleic acids, polynucleotide molecules, vectors, and the like into a target cell(s), and more specifically into the interior of a membrane-enclosed space of a target cell(s).
  • the molecules can be "introduced” into the target cell(s) by any means known to those of skill in the art, for example as taught by Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001), the contents of which are incorporated by reference herein.
  • Means of "introducing" molecules into a cell include, for example, but are not limited to: heat shock, calcium phosphate transfection, PEI transfection, electroporation, lipofection, transfection reagent(s), viral-mediated transfer, and the like, or combinations thereof.
  • the transfection of the cell may be performed on any type of cell, of any origin, such as, for example, human cells, animal cells, plant cells, virus cell, and the like.
  • the cells may be selected from isolated cells, tissue cultured cells, cell lines, cells present within an organism tissue and body, and the like.
  • treating and “treatment” as used herein refers to abrogating, inhibiting, slowing or reversing the progression of a disease or condition, ameliorating clinical symptoms of a disease or condition or preventing the appearance of clinical symptoms of a disease or condition.
  • preventing is defined herein as barring a subject from acquiring a disorder or disease or condition.
  • treatment of cancer is directed to include one or more of the following: a decrease in the rate of growth of the cancer (i.e. the cancer still grows but at a slower rate); cessation of growth of the cancerous growth, i.e., stasis of the tumor growth, and, the tumor diminishes or is reduced in size.
  • the term also includes reduction in the number of metastases, reduction in the number of new metastases formed, slowing of the progression of cancer from one stage to the other and a decrease in the angiogenesis induced by the cancer. In most preferred cases, the tumor is totally eliminated. Additionally included in this term is lengthening of the survival period of the subject undergoing treatment, lengthening the time of diseases progression, tumor regression, and the like.
  • the cancer is a solid tumor.
  • the cancer is breast cancer.
  • Metastasis As used herein, the terms “metastasis”, “cancer metastasis” or “tumor metastasis” are used interchangeably and refer to the growth of cancerous cells derived from the primary cancerous tumor in another location or tissue. Metastasis also encompasses micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part which is not directly connected to the organ of the original, primary cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from an original tumor site, and migration and/or invasion of cancer cells to other parts of the body.
  • reducing or preventing cancer metastasis refers to slowing or even completely inhibiting the formation, spread, development and/or growth of metastasis from the primary tumor.
  • the term may also include reducing the number of metastases in an organ or tissue, as well as reducing the size and/or malignancy status of existing metastases.
  • organism refers to a mammal. In some embodiments, the organism is human. In some embodiments, the organism is selected from a pet, a rodent, a farm animal, and a lab animal.
  • the term "subject" is interchangeable with an individual or patient.
  • the subject is a mammal.
  • the subject is a human.
  • the subject is symptomatic.
  • the subject is asymptomatic.
  • the subject is a human afflicted with cancer.
  • the subject is preferably an individual with the C allele of the polymorphic site within the PALLD gene identified by reference number rsl071738.
  • the subject is at risk of developing metastasis.
  • the subject has already developed metastases.
  • small interfering RNA and “siRNA” are used interchangeably and refer to a nucleic acid molecule mediating RNA interference or gene silencing.
  • the siRNA inhibits expression of a target gene and provides effective gene knock-down.
  • microRNA and "miRNA” are directed to a small non-coding RNA molecule that can function in transcriptional and post-transcriptional regulation of target gene expression.
  • the terms encompasses a mature miRNA sequence or a precursor miRNA sequence, including a primary transcript (pri-miRNA) and a stem-loop precursor (pre-miRNA).
  • pri-miRNA primary transcript
  • pre-miRNA stem-loop precursor
  • the biogenesis of a miRNA initiates in the nucleus by RNA polymerase II transcription, generating a primary transcript (pri- miRNA).
  • the primary transcript is cleaved by Drosha ribonuclease III enzyme to produce an approximately 70 nt stem-loop precursor miRNA (pre-miRNA).
  • the pre-miRNA is then actively exported to the cytoplasm where it is cleaved by Dicer ribonuclease to form the mature miRNA.
  • One strand of this miRNA is incorporated into an RNA-induced silencing complex (RISC) which recognizes target mRNAs through imperfect base pairing with the miRNA, and most commonly results in translational inhibition or destabilization of the target mRNA.
  • RISC RNA-induced silencing complex
  • the target mRNA contains a sequence complementary to a "seed" sequence of the miRNA, which usually corresponds to nucleotides 2-8 of the miRNA.
  • the seed sequence is considered to be essential for the binding of the miRNA to the mRNA.
  • miRNA databases such as miRBase (Griffiths- Jones et al. 2008 Nucl Acids Res 36, (Database Issue: D154-D158) and the NCBI human genome database.
  • miRNA molecules and “miR molecules” refer to the miR-96 and/or miR-182 miRNA molecules. When referring to the miR molecules, the reference is to either one or both of said miRNA molecules. Each possibility being a separate embodiment.
  • polynucleotides of the invention refers to the miRNA molecules (i.e. miR-96 and/or miR-182) and/or to vectors expressing or encoding the same.
  • the miRNA molecules may be modified at the base moiety, sugar moiety, or phosphate backbone, for example, in order to improve stability of the molecule, hybridization, transport into the cell, and the like. In addition, modifications can be made to reduce susceptibility to nuclease degradation.
  • the miR molecules may have other appended groups such as peptides (for example, for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane or the blood-brain barrier, hybridization-triggered cleavage agents or intercalating agents.
  • Various other well known modifications can be introduced as a means of increasing intracellular stability and half- life.
  • nucleic acids having modified internucleoside linkages such as 2'-0-methylation may be used.
  • Nucleic acids containing modified internucleoside linkages may be synthesized using reagents and methods that are well known in the art.
  • the term "plurality" as used herein is directed to include more than one component.
  • the term "about”, when referring to a measurable value is meant to encompass variations of +/-10 , more preferably +1-5%, even more preferably +/-1 , and still more preferably +/-0.1 % from the specified value.
  • a method for reducing or preventing cancer metastasis in a subject in need thereof comprising administering to the subject at least one miRNA molecule selected from the group consisting of miR-96 and miR-182, or at least one vector expressing or encoding the same, thereby reducing or preventing cancer metastasis in the subject.
  • a method for reducing or preventing cancer metastasis cancer metastasis in a subject in need thereof comprising administering to the subject miR-96 and/or miR-182, or a corresponding vector expressing or encoding the same, thereby reducing or preventing cancer metastasis in the subject.
  • a method of reducing or preventing cancer metastasis in a subject in need thereof include inhibiting or reducing expression of palladin in the cancer cells and/or cancer metastasis cells.
  • reducing or inhibiting expression of palladin is achieved by administration of miR-96 and/or-miR- 182 or one or more vectors encoding for said miRNA molecules.
  • miR-96 and/or-miR- 182 or one or more vectors encoding for said miRNA molecules are examples of a separate embodiment.
  • a method for treating cancer in a subject in need thereof comprising administering to the subject at least one miRNA molecule selected from the group consisting of miR-96 and miR- 182, or at least one vector expressing or encoding the same, thereby treating cancer in the subject.
  • a method for treating cancer in a subject in need thereof comprising administering to the subject miR-96 and/or miR-182, or a corresponding vector expressing or encoding the same, thereby treating cancer in the subject.
  • a method of treating cancer in a subject in need thereof include inhibiting or reducing expression of palladin in the cancer cells.
  • reducing or inhibiting expression of palladin is achieved by administration of miR-96 and/or-miR- 182 or one or more vectors encoding for said miRNA molecules.
  • the methods may further include determining that the subject is carrying the C allele of the single nucleotide polymorphism (SNP) rsl071738 prior to administering the at least one miRNA molecule or the at least one vector expressing or encoding the same.
  • SNP single nucleotide polymorphism
  • the subject is at risk of developing metastasis and the administering is carried out prior to metastasis formation. In some embodiments, the subject has already developed metastases and the administering is carried out after metastasis formation.
  • the cancer is a cancer associated with an abnormal palladin expression and/or activity.
  • a pharmaceutical composition comprising at least one miRNA molecule selected from the group consisting of miR-96 and miR-182, or at least one vector expressing or encoding the same, for use in reducing or preventing cancer metastasis.
  • a pharmaceutical composition comprising miR-96 and/or miR-182, or corresponding vector(s) expressing or encoding the same, for use in reducing or preventing cancer metastasis.
  • a pharmaceutical composition comprising at least one miRNA molecule selected from the group consisting of miR-96 and miR-182, or at least one vector expressing or encoding the same, for use in treating cancer.
  • a pharmaceutical composition comprising miR-96 and/or miR-182, or corresponding vector(s) expressing or encoding the same, for use in treating cancer.
  • a pharmaceutical composition comprising miR-96 and/or miR-182, or corresponding vector(s) expressing or encoding the same, for use in reducing palladin expression in cancer cells.
  • each of miR-96 and miR-182, or the corresponding vectors expressing or encoding the same are formulated in distinct compositions (such as pharmaceutical compositions), that may be administered concomitantly or separately.
  • both the miR-96 and the miR-182, or the corresponding vectors expressing or encoding the same are formulated in one composition (such as pharmaceutical composition).
  • both the miR-96 and miR-182 are encoded or expressed by the same vector. In some embodiments, each of the miR-96 and the miR-182 are encoded or expressed by separate vectors.
  • use of a composition and/or treatment according to the present invention is carried out through direct provision of miRNA.
  • treatment according to the present invention is carried out through introduction of an expression vector containing DNA encoding the miRNA.
  • treatment according to the present invention is carried out through introduction of a vector containing expressing the miRNA.
  • the miRNA, or DNA sequence encoding the same may include mature miRNA sequences, pri-miRNA sequences or pre-miRNA sequences.
  • use of dedicated delivery platforms may be utilized, for a specific and efficient delivery of the miRNA molecules to target cells and organs, in- vivo.
  • a mature miR-96 comprises or consists of nucleotide sequence: UUUGGCACUAGCACAUUUUUGCU (SEQ ID NO: 1).
  • an exemplary pri-miRNA of miR-96 comprises or consists of nucleotide sequence:
  • an exemplary pre-miRNA of miR-96 comprises or consists of nucleotide sequence:
  • a mature miR- 182 comprises or consists of nucleotide sequence: UUUGGCAAUGGUAGAACUCACACU (SEQ ID NO: 4).
  • an exemplary pri-miRNA of miR- 182 comprises or consists of nucleotide sequence:
  • an exemplary pre-miRNA of miR- 182 comprises or consists of nucleotide sequence:
  • RNA sequences containing U nucleotides are RNA sequences containing U nucleotides. It is to be understood that when expression vectors are used, containing DNA sequences encoding the RNA, T nucleotides should be provided.
  • analogues of the miRNA sequences provided herein may be used, as long as they maintain the ability to regulate their target.
  • "Analogs" herein encompass miRNA sequences of the present invention in which one or more bases are substituted or deleted.
  • the "seed" sequence which is a sequence completely complementary to a sequence within the mRNA targeted by the miR and thus essential for the binding, should remain unchanged.
  • the seed sequence corresponds to positions 2-7 of the miR.
  • the miR molecule may be designed to match the mutated sequence.
  • the miR may be designed with a nucleotide matching the mutated one on the target sequence, i.e., a nucleotide complementary to the mutated one.
  • an analogue has at least about 75% identity or complementarity to a sequence of the invention, for example at least about 80%, at least about 85%, at least about 90%, at least about 99% identity or complementarity to a sequence of the invention.
  • Each possibility represents a separate embodiment of the present invention.
  • the miR molecules may be introduced to a cell, a tissue or an organism by any of the methods known in the art.
  • the miR molecules may be introduced in the form of a composition (one composition or separate compositions).
  • the composition is a pharmaceutical composition, comprising one or more suitable excipients.
  • the miR molecules may be expressed or encoded in a target cell, tissue or organism by an exogenous vector introduced thereto.
  • the vector may be comprised in a composition.
  • the vector may be introduced to a cell, tissue or organism by any of the methods known in the art.
  • the miRNA molecules may be introduced in the form of a single strand RNA molecule (ssRNA), double strand RNA molecule (dsRNA), or an RNA molecule which is at least partially double stranded. Each possibility is a separate embodiment.
  • the vector miRNA molecules may be encoded by one vector. In some embodiments, the miRNA molecules may be encoded by separate vectors.
  • the miR-96 and the miR- 182 may be encoded by a single vector. In some embodiments, the miR-96 and the miR- 182 may each be encoded by a separate vector. In some embodiments, the miR-96 and the miR- 182 may be formulated in the same composition or in separate compositions.
  • the miR-molecules may be introduced or expressed or encoded in a cell, tissue or organism in combination with one or more additional reagent.
  • the additional reagent may be a therapeutic reagent (drug).
  • the additional reagent may include other polynucleotide molecule(s).
  • the miR-molecules (or vector(s) encoding or expressing the same) and the additional reagent may be administered in the same or different composition and they may be administered simultaneously, or sequentially, at any time interval.
  • the miRNA molecules or vectors encoding the same may be administered systemically (enterally or parenterally) or locally, for example via intra- tumor injection. Each possibility represents a separate embodiment of the present invention.
  • administration is systemic administration. In other embodiments, the administration is localized administration. In some embodiments, localized administration is into a tumor. In additional embodiments, localized administration is into a space or cavity adjacent to a tumor. In other embodiments, localized administration is into a space or cavity formed after tumor resection.
  • Non- limiting examples of suitable administration routes include intravenous, intramuscular, subcutaneous, transdermal, intradermal and oral administration. Each possibility represents a separate embodiment of the present invention.
  • miRNA molecules or expression vectors are injected into a space adjacent to a tumor. In other embodiments, they are injected into a space formed following excision of the tumor.
  • targeting moieties may be used.
  • Targeting the nucleic acid constructs to a particular cell can be performed by any method known to those skilled in the art.
  • the construct can be conjugated to an antibody that recognizes cell surface antigens unique to cancer cells, or that are more prevalent on cancer cells, compared to normal cells.
  • the construct can be conjugated to a ligand specifically recognized by receptors unique to cancer cells, or that are more prevalent on cancer cells.
  • various delivery systems are known and can be used to transfer/introduce the polynucleotides and/or composition of the invention into cells, such as, for example, encapsulation in liposomes, targeted liposomes, dendtritic polyglycerolamine nanocarriers, nanoparticles, microparticles, microcapsules, electroporation, nucleofection, ultrasound based, laser based, recombinant cells that are capable of expressing the composition, receptor-mediated endocytosis, construction of the composition of the invention as part of a viral vector or other vector, viral vectors that are capable of being reproduced without killing the cell during the process of reproduction and that comprise the composition of the invention, viral vectors that are not capable of reproduction and that comprise the composition of the invention, injection of cells that produce viral vectors that comprise the composition of the invention, injection of polynucleotides, electroporation, calcium phosphate mediated transfection, and the like, or any other methods known in the art or to be developed in the
  • the polynucleotide and compositions of the invention may be suitably formulated for intravenous, intramuscular, subcutaneous, intracervical, intratumoral, or intraperitoneal administration.
  • the polynucleotide and compositions described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • formulations for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the polynucleotides and composition of the invention can be administered by any convenient protocol.
  • the protocol employed is a nucleic acid administration protocol, where a number of different such protocols are known in the art.
  • the nucleic acids may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, fusion of vesicles, or Jet injection for intramuscular administration.
  • the nucleic acids may be coated onto gold microp articles, and delivered intradermally by a particle bombardment device.
  • expression vectors may be used to introduce the nucleic acids into a cell.
  • the polynucleotides or compositions of the invention may be fed directly to, injected into, the host organism containing a desired target gene.
  • the polynucleotides or compositions of the invention may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, and the like.
  • Methods for oral introduction include direct mixing of a polynucleotide (such as, RNA) with food of the organism.
  • Physical methods of introducing polynucleotides include injection directly into the cell or extracellular injection into the organism of a polynucleotide solution (composition), such as, an RNA solution.
  • the polynucleotides of the invention may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (for example, at least 5, 10, 100, 500 or 1000 copies per cell) of the polynucleotide may yield an enhanced effect, whereas lower doses may be useful for specific applications. In some embodiments, a hydrodynamic nucleic acid administration protocol may be used. In some embodiments, the polynucleotides of the invention can be incorporated into a variety of formulations (compositions) for therapeutic administration.
  • the polynucleotides of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi- solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, intratumoral, intracervical, intra- tissue and the like, administration.
  • the polynucleotides may be administered alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the pharmaceutical dosage forms may be administered locally, by being disposed or contained in a device.
  • use of dedicated delivery platforms may be utilized, for a specific and efficient delivery of the miRNA molecules to target cells and organs, in- vivo.
  • any suitable devilry vehicle may be used to deliver the polynucleotides of the invention to target cell, tissue or organ (such as cancer cell and/or metastases).
  • the delivery is specific, efficient and targeted, such that non-target cells, tissues or organs are not affected.
  • nanocarriers may be used to deliver the polynucleotides of the invention.
  • the nanocarriers may include dendritic polyglycerolamine (dPG-NH2).
  • dPG-NH2 is a cationic hyperbranched polymer, that can improve miRNA and siRNA stability, intracellular trafficking, silencing efficacy, and accumulation in the tumor environment due to the enhanced permeability and retention effect.
  • dPG-NH2 exhibited low cytotoxicity and high efficacy in delivering active siRNA/miRNA into cells.
  • liposomal particles such as, targeted liposomes may be used to deliver the polynucleotides of the invention.
  • targeted liposomes that can deliver polynucleotide molecules (such as, miRNAs) may be targeted using an appropriate targeting moiety, such as antibodies targeting specific cancer cells, for example, based on receptors present on these cells.
  • the particles may be coated with glycosaminoglycan (such as, hyaluronan).
  • nanoparticles, coupled to pentapeptide may be used to deliver the polynucleotides of the invention.
  • the pentapeptide coupled carriers utilize the pentapeptide Tyr-Ile-Gly-Ser-Arg (YIGSR) (SEQ ID NO: 17), that can enhance specific binding to cancer cells and cancer metastatic cells, thereby efficiently and specifically deliver the polynucleotides of the invention to target cells, tissues and organs.
  • the dosage and frequency of administration may be selected in relation to the pharmacological properties of the nucleic acids to be delivered (i.e., naked RNA, vectors, delivery particles used, and the like).
  • the miR molecules (alone or in combination with other agents) may be administered in a dose having an amount of between about O.Olmg and about lOmg per administration/treatment per day/per miRNA molecules.
  • the amount may be between about 0.01 mg and about 8mg per administration/treatment.
  • the amount may be between about O.Olmg and about 2mg per administration/treatment.
  • the amount may be between about 0.05mg and about 4mg per administration/treatment.
  • the amount may be between about 0.05mg and about 2mg per administration/treatment.
  • the amount may be between about 0.08mg and about 2mg per administration/treatment.
  • the amount may be between about 0.08mg and about lmg per administration/treatment.
  • the amount may be between about 0.5mg and about 9mg per administration/treatment.
  • the miR molecules may be formulated in a saline solution (such as PBS).
  • the doses disclosed herein may be administered at any administration regime, such as, 1-5 times a day; 1-10 times a week, 1-15 times a month, and the like, at identical or different time intervals and/or at the same or different time of day.
  • the polynucleotides can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, polyglutamic acid (PLGA) poly lysine acid (PLA), corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, polyglutamic acid (PLGA) poly lysine acid (PLA), corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or ge
  • the polynucleotides can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
  • compositions of the invention may be advantageously combined and/or used in combination and/or alternation with other agents which are either therapeutic or prophylactic agents, and different from the subject compounds.
  • the compositions may also be advantageously combined and/or used in combination with agents that treat conditions often associated with the treated condition.
  • administration in conjunction with the subject compositions enhances the efficacy of such agents.
  • the therapeutic agents may include chemotherapeutic agents, such as, Alkylating agents, Anthracyclines, Cytoskeletal disrupters Taxanes), Epothilones, Histone Deacetylase Inhibitors, Inhibitors of Topoisomerase I, Inhibitors of Topoisomerase II, Kinase inhibitors, Nucleotide analogs and precursor analogs, Peptide antibiotics, Platinum-based agents, Retinoids, Vinca alkaloids and derivatives, and the like, or combinations thereof.
  • chemotherapeutic agents such as, Alkylating agents, Anthracyclines, Cytoskeletal disrupters Taxanes), Epothilones, Histone Deacetylase Inhibitors, Inhibitors of Topoisomerase I, Inhibitors of Topoisomerase II, Kinase inhibitors, Nucleotide analogs and precursor analogs, Peptide antibiotics, Platinum-based agents, Retinoids, Vinca alkaloids and derivatives, and the like, or combinations thereof.
  • the delivery vehicle of the nucleic acid constructs comprises a targeting moiety or a mechanism for selective activity within the cancer cells only.
  • cancers that can be treated by the compositions and methods disclosed herein include such cancers as: carcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed type tumors.
  • Particular categories of tumors include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, lung cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above.
  • tumors amenable to treatment include: hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyo sarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), renal cell carcinoma, hypernephroma, hypemephroid adenocarcinoma, bile duct carcinoma, choriocar
  • the cancer is selected from cervical cancer, hepatic cancer, prostate cancer, breast cancer, skin cancer, colon cancer, lung cancer, pancreatic cancer, lymphoma, myeloma, leukemia, head and neck cancer, kidney cancer, ovarian cancer, bone cancer, liver cancer or thyroid cancer.
  • cervical cancer hepatic cancer
  • breast cancer breast cancer
  • colon cancer lung cancer
  • pancreatic cancer lymphoma
  • myeloma leukemia
  • head and neck cancer kidney cancer
  • ovarian cancer bone cancer
  • liver cancer or thyroid cancer thyroid cancer
  • the cancers to be treated by the compositions and methods of the present invention are solid tumors, preferably solid tumors in which abnormal palladin activity and/or expression is associated with their pathogenesis, e.g., promotes invasiveness of the cancer.
  • Such cancers include, e.g., cancers in which palladin is over- expressed and/or mutated.
  • the cancer is selected from the group consisting of breast cancer, pancreatic cancer and colorectal cancer. Each possibility represents a separate embodiment of the present invention.
  • the cancer is breast cancer.
  • reduction or prevention of metastasis development can be measured by standard methodologies known in the art including a reduction in size or numbers of tumors as measured by a variety of radiographic, imaging, circulating tumor marker, palpitation, direct measurement or observation techniques known in the art. Accordingly a reduction or prevention of metastasis development can also be measured by a reduction of a sign or symptom associated with the disease state of the cancer being treated or a prolongation of survival or reduction in suffering from a disease sign or symptom of the cancer being treated.
  • kits thereof for practicing one or more of the above-described methods.
  • the subject reagents and kits thereof may vary greatly.
  • the kits at least include mir-96 and/or miR-182 molecule or vector(s) encoding or expressing the same, as described above.
  • the kits may also include a pharmaceutically acceptable delivery vehicle, which may be combined with or separate from the miR-molecules in the kit.
  • the kits further include instructions for practicing the subject methods.
  • kits comprising the pharmaceutical composition, essentially as described above, and instructions for use of the kit.
  • nucleic acids of the invention when treating cancer, administration of the nucleic acids of the invention (i.e., miRNA molecules or vectors(s) encoding or expressing the same), may be performed in combination with one or more additional treatments.
  • combination therapy may be used to increase tumor susceptibility to chemotherapy and/or irradiation.
  • repeated administration of the nucleic acids of the invention may be performed, wherein the dosages administered and the composition of the nucleic acid may be identical, similar or different.
  • the administration may be prolong (such as over the course of 1-120 hours).
  • the term comprising includes the term consisting of.
  • miRs The micro-RNAs (“miRs”) miR-96 and miR-182 were identified as potential regulators of palladin protein expression in humans.
  • PALLD Fragments of the 3 ' UTR of PALLD spanning the miRNA binding sites were cloned downstream to the Renilla Luciferase Reporter of the psiCHECKTM-2 plasmid (Promega) that contains a Firefly Luciferase Reporter (used as control) under a different promoter.
  • the PALLD gene has a known G/C polymorphism within the binding site of miR-96 and miR-182 (refSNP - rsl071738, see dbSNP website).
  • Pre-miRNAs human miR-182 and miR-96 were cloned into the miRNA expression vector miRVec.
  • HEK-293T or HeLa cells were co-transfected with the psiCHECKTM-2 containing the desired 3' UTR and miRVec containing the desired pre- miRNA.
  • Firefly and Renilla Luciferase activities were measured using the Dual-Luciferase Reporter Assay System kit (Promega) and a Veritas microplate luminometer.
  • Example 2 Levels of endogenous palladin in cells over-expressing miR-96/miR-182
  • Hs578 cells human mammary carcinoma
  • miR-96 and miR-182 were tested by measuring endogenous levels of palladin mRNA and protein in Hs578 cells (human mammary carcinoma) transfected with either miR-96 or miR-182 (expressed from miRvec plasmid as mentioned above).
  • the Hs578 cells are heterozygote in the polymorphic position within the binding site of the miRs, having one C allele and one G allele.
  • Hs578 cells were transfected when cells were 60-75% Confluent. DNA plasmids were transfected together with a transfection reagent (lipofectamin 2000, Invitrogen) in Optimem serum (Biological Industries). GFP was transfected as a control and its detection was confirmed 24 hrs after transfection. 24hrs following transfection the cells were harvested for RNA and protein extractions. Measuring mRNA and miRNA levels: Total RNA was extracted using TRIzol reagent (Invitrogen, Life Technologies) and RNA quality was measured using a NanoDrop (Thermo Scientific). cDNA for miRNA and mRNA was synthesized from total RNA.
  • Reverse transcription reaction for mRNA was carried out using random-primer and Superscript III reverse transcriptase (Invitrogen). Reverse transcription reaction for specific miRNA was done using TaqMan miRNA Assays (Applied Biosystems; ABI). Single miRNAs/mRNAs expression were tested similarly using TaqMan Universal PCR Master Mix (No AmpErase UNG; Applied Biosystems) or SYBR green PCR master mix (Applied Biosystems), respectively, using Step-One Sequence Detection System. Expression values were calculated based on the comparative threshold cycle (Ct) method. MiRNAs levels were normalized to U6 and mRNA expression levels were normalized to GAPDH.
  • Ct comparative threshold cycle
  • Hs578 cells were homogenized with a lysis buffer. Protein levels in the lysates were determined by using the Bio-Rad protein assay (Bio- Rad). Lysates were resolved by SDS-PAGE through 4-12% gels (GeBaGel, Gene Bio- Application) and transferred by electroporation to nitrocellulose membranes. Membranes were blotted with anti-palladin (Protein Group) or anti-actin (Millipore) antibodies, followed by a secondary antibody linked to horseradish peroxidase. Band quantification was performed using ImageJ software (National Institutes of Health).
  • 4T1 cells (mice mammary carcinoma) which are homozygote to the C allele in the miRs' binding site were stably transformed with mouse miR-96 ("mmu-miR-96”), mouse miR-182 ("mmu-miR-182”) or a scrambled sequence, and the levels of palladin protein were measured.
  • a CD515B plasmid expressing the scrambled sequence under a CMV promoter was used as a control.
  • Pre-miR-182 and pre- miR-96 of mice were amplified by PCR (using 4T1 cells' DNA as template) and each cloned into a CD515B plasmid, under the CMV promoter.
  • the sequences of the three plasmids, following the CMV promoter, are:
  • mmu-miR-182 mature sequence TTTGGCAATGGTAGAACTCACACCG (SEQ ID NO: 9)
  • mmu-miR-96 mature sequence TTTGGCACTAGCACATTTTTGCT (SEp ID NP: 12)
  • sequences marked in boldface correspond to the recognition site of the restriction enzyme Xbal; underlined sequences correspond to the recognition site of Nhel; sequences shown in italics correspond to the recognition site of EcoRI; sequences marked in boldface and underlined correspond to the recognition site of Notl.
  • Retroviral particles were prepared using HEK-293 cells that were co-transfected with CD515B plasmids and lentiviral vector packaging (Tarom) using PEI transfection reagent (sigma). Forty-eight hours following transfection retroviral particles were collected. For infection, the retroviral particles containing medium was added to 4T1 cells at 50% confluence in 6 wells plates. 48 hour later, Hygromycin (200ug/ml, Sigma) was added to the medium for selection.
  • Hs578 cells were plated in 12-well plates, transfected as indicated, and cultured to confluency. Cells were serum-starved for 8h and scraped with a P200 tip (time 0). The percentage of open wound was assessed from pictures (five fields) taken at the indicated time points, using ImageJ software. The results are shown in Figure 4A.
  • Invasion - Matrigel invasion assay The invasive potential was assessed using Matrigel invasion chambers (BD Biosciences).
  • One chamber consists of a cell insert and a well. The bottom of the cell insert is covered with a filter containing multiple 8-mm pores and is coated with a basement membrane matrix (Matrigel).
  • Hs578 transfected cells were serum starved O/N (starvation started 24h following transfection for Hs578), harvested and re- suspended in serum free medium. Hs578 (5*10 4 ) were plated in serum-free medium in transwell inserts. Complete medium served as chemo-attractant in the lower chamber.
  • MCF7 cells human mammary carcinoma
  • MCF7 cells were transfected when cells were 60-75% Confluent.
  • Anti-miR miRNA inhibitors (Ambion (cat# AM17000)) for hsa-miR-182 and hsa-miR-96 or a scrambled sequence (mirVanaTM miRNA Inhibitor, Negative Control #1, Cat. number: 4464076) were transfected together with a transfection reagent (lipofectamin 2000, Invitrogen) in Optimem serum (Biological Industries). GFP was transfected as control and its detection was confirmed 24 hrs after transfection. 24hrs following transfection the cells were harvested for RNA extraction.
  • MCF7 cells were plated in 12-well plates, transfected as indicated, and cultured to confluency. Cells were serum-starved for 8h and scraped with a P200 tip (time 0). The percentage of open wound was assessed from pictures (five fields) taken at the indicated time points, using ImageJ software. The results are summarized in Figure 5D (left panel - pictures taken at the indicated time points following scrape; right panel - percentage of open wound at each time point compared to time 0). Down-regulation of miR-96 or miR-182 resulted in increased cell migration.
  • Palladin mRNA and protein levels following stable over-expression of palladin shRNA were assayed.
  • 4T1 cells were infected with palladin shRNA or a scrambled shRNA as control (Palladin shRNA target sequence: GCTAACCTATGAGGAAAGAAT (SEQ ID NO: 18)).
  • Palladin shRNA target sequence GCTAACCTATGAGGAAAGAAT (SEQ ID NO: 18)
  • palladin mRNA was down-regulated in the palladin shRNA- infected cells (palladin knock-down (KD) cells) compared to cells infected with the control sequence (CCGGGCGCGATAGCGCTAATAATTTCTCGAGAAATTATTAGCGCTATCGCGCT TTTT (SEQ ID NO: 19)).
  • palladin protein was extracted from the cells and the level of palladin protein (isoform 4, 90kDa) was evaluated by Western blot (Figure 6B, left panel). Bands quantification was done using ImageJ software and protein levels were normalized to Actin levels ( Figure 6B, right panel). Stable over-expression of palladin shRNA resulted in decreased palladin protein levels.
  • Example 7 Effect of miR-96/miR-182 on tumor metastasis in vivo
  • the 4T1 mammary carcinoma is a transplantable tumor cell line.
  • the tumor is typically grown in BALB/c mice and in tissue culture, and is highly tumorigenic and invasive. In addition, it can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone.
  • 4T1 cells constitutively expressing mCherry fluorescent protein and stably transformed with miR-96, miR-182 or a scrambled RNA were used.
  • the constitutive expression of mCherry enables monitoring tumor growth and metastases spread in the mice, using an imaging system (CRI-MAESTROTM).
  • mice Female BALB/c mice (5-6 weeks old) were weighted and anesthetized by intraperitoneal (i.p) injection of 150ul Ketamine-Xylazin solution. Next, 100ul 4Tl cells (lxlO 6 cells) were injected subcutaneously into the mammary gland. Each group of mice (tumor cells over-expressing miR-96, miR-182 or a scrambled RNA) contained 6 mice.
  • Tumor onset was monitored daily by palpating the injection area for the presence of a tumor. Mice weight and tumor size were checked every 3 to 4 days starting from day 5. Tumor size was determined by measuring two perpendicular measurements of the tumor (width and length) using Vernier caliper. The mean tumor diameter (TD) and the tumor volume (TV) were calculated.
  • Primary tumor removal Primary tumors were surgically removed when tumor measurements reached about 10x10 mm (lengthX width) or when tumors become necrotic. Prior to removal, the tumors were imaged using CRI-MAESTROTM imaging system in order to measure fluorescent signals from the tumor and determine tumor area based on the fluorescence measurements. The removed tumors were weighted and stored in (-80)°C for farther analyzes. 6. Monitoring mice survival. Mice survival was monitored following tumor removal. The survival of the mice from the tumor inoculation date and the tumor removal date were tracked.
  • mice were weighted and anesthetized. The recurrence of the primary tumor was checked by imaging the primary tumor area using CRI-MAESTROTM imaging system. If a fluorescent signal was detected at the primary tumor area, measurements of total fluorescent signal and tumor area were taken.
  • CT computed tomography
  • the primary tumors from mice injected with 4T1 cells stably transformed with either miR-96, miR- 182 or a scrambled RNA molecule were analyzed for the levels of the respective miR, palladin mRNA and palladin protein. Following removal, all tumors were cut into a few pieces. One piece of each primary tumor was placed in an eppendorf tube containing Igepal lysis buffer for protein extraction, and another piece was placed in another eppendorf tube containing Trizol for RNA extraction. Tumors homogenization was done using TissueLayser (Qiagene). Measuring miRNA, mRNA and protein levels were done as mentioned above (example 2, pages 2-3).
  • Figures 9A-C summarize measurements of tumor volume (Figure 9A), tumor diameter ( Figure 9B) and mice weight ( Figure 9C) in each group of mice starting from day 5 following inoculation of the tumor cells.
  • Figure 9D shows exemplary fluorescence measurements of primary tumors from each group of mice at the removal day of the tumors.
  • the upper panel of each group shows the total signal, and the lower panel shows the signal from the tumor area.
  • Figure 9E shows the average tumor area of the primary tumors at their removal day in each group of mice, as determined from the fluorescence measurements. The average weights of the primary tumors at the day of their removal in each group are shown in Figure 9F.
  • Figure 10A shows exemplary CT photos of lungs from mice in each group.
  • Metastatic nodules are marked with an arrow.
  • Figures lOB-C show the average quantity of lung metastatic nodules detected in the CT photos and the average diameter of lung metastases in each group of mice (no metastasis were detected in the brain or liver).
  • Figures 10D-E show exemplary fluorescence measurements in lungs and the average percentage of fluorescent area per lung in each group of mice. Metastatic areas are circled with white line.
  • Example 8 In-vivo delivery of miR-96/miR-182 using delivery platforms
  • Dendritic polyglycerolamine (dPG-NH?) nanocarriers- dPG-NH2 is a cationic hyperbranched polymer, which was shown to improve miRNA and siRNA stability, intracellular trafficking, silencing efficacy, and accumulation in the tumor environment due to the enhanced permeability and retention effect (Ofek P., et al. In vivo delivery of small interfering RNA to tumors and their vasculature by novel dendritic nanocarriers.
  • dPG-NH2 was shown to exhibit low cytotoxicity and high efficacy in delivering active siRNA/miRNA into cells.
  • 4T1 mouse breast cancer cells are inoculated into the mammary fat pad of BALB/c female mice (BALB/cAnNCrl). Once palpable tumors develops, 10 mg/kg dPG-NH2 complexed with 4 mg/kg miR-96/182, NC-miR, or PBS is injected intratumorally every 3 days. Primary tumors are removed seven days after the first dPG- NH2-miRNA polyplex injection. The presence of metastases in the lungs is evaluated by micro-CT for additional twenty-one (21) days. Then, mice are sacrificed and the harvested organs harvested (lungs, liver and brain) are screened for the presence of macro- metastases.
  • Targeted liposomes - Targeted liposomes encapsulating small RNAs are used, in combination with, or separately from other treatments. It was shown that stabilized liposomes can deliver siRNAs into leukocytes involved in gut inflammation to inhibit colitis in a mouse model (Peer, D., et al. Systemic leukocyte-directed siRNA delivery revealing cyclin Dl as an anti- inflammatory target. Science 2008 ; 319:5863).
  • Antibodies targeting specific cancer cells are used as targeting moieties for delivering our the miRNAs molecules specifically to target cenacer cells and metastates, in order to effectively reduce the metastatic potential of the cells.
  • each liposome carries a high payload (-4000 siRNAs/miRNAs per particle), allowing therapeutic efficacy at a low dose (-2.5 mg/kg).
  • the liposomal particles are coated with hyaluronan, a naturally occurring glycosaminoglycan that stabilizes small RNA entrapment, inhibits nonspecific uptake in vivo, and serves as the attachment site for the selected antibody. The effect of the treatments using these targeted liposomes on the tumor and metastasis is evaluated as described above.
  • Pentapeptide-coupled nanoparticles - nanoparticles which carry the microRNA molecules, which are coupled to a pentapeptide which enhances binding to cancer cells and cancer metastatic cells are utilized.

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Abstract

L'invention concerne des compositions et des méthodes pour le traitement du cancer, en particulier des tumeurs solides et des métastases cancéreuses, faisant appel aux micro-ARN miR-96 et/ou miR-182.
PCT/IL2016/050759 2015-07-15 2016-07-14 Micro-arn pour le traitement des tumeurs solides malignes et des métastases WO2017009837A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017223492A1 (fr) * 2016-06-23 2017-12-28 Massachusetts Institute Of Technology Compositions, dispositifs, et procédés d'administration d'arnmicro
CN107881239A (zh) * 2017-12-27 2018-04-06 广西壮族自治区肿瘤防治研究所 血浆中与结直肠癌转移相关的miRNA标志物及其应用
CN108004323A (zh) * 2017-12-27 2018-05-08 广西壮族自治区肿瘤防治研究所 组织中与结直肠癌转移相关的miRNA标志物及其应用
WO2020002565A1 (fr) 2018-06-27 2020-01-02 Sabine Bauer Implants pour recruter et retirer des cellules tumorales circulantes
WO2021122803A2 (fr) 2019-12-17 2021-06-24 Cirlo Gmbh Ensembles dispositifs de cathéter allongés de forme tubulaire destinés à entrer en interaction avec des composants de fluides corporels, procédé de récupération de cellules, d'agrégats de cellules et d'exosomes à partir d'un dispositif de cathéter allongé de forme tubulaire et ensembles dispositifs de cathéter allongés de forme tubulaire intelligents pour surveiller l'interaction avec des composants de fluides corporels

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010056737A2 (fr) 2008-11-11 2010-05-20 Mirna Therapeutics, Inc. Procédés et compositions impliquant des miarn dans des cellules souches cancéreuses
WO2010061396A1 (fr) * 2008-11-30 2010-06-03 Rosetta Genomics Ltd. Procédés pour détecter une susceptibilité augmentée au cancer
US20110021607A1 (en) 2008-02-01 2011-01-27 Michael Clarke Methods and Compositions Relating to Carcinoma Stem Cells
WO2012005572A1 (fr) * 2010-07-06 2012-01-12 Interna Technologies Bv Miarn et ses utilisations diagnostiques et thérapeutiques pour des maladies ou des états associés au mélanome, ou pour des maladies ou des états associés à la voie braf activée
US20140088170A1 (en) 2012-08-29 2014-03-27 City Of Hope Differentially expressed microrna molecules for the treatment and diagnosis of cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110021607A1 (en) 2008-02-01 2011-01-27 Michael Clarke Methods and Compositions Relating to Carcinoma Stem Cells
WO2010056737A2 (fr) 2008-11-11 2010-05-20 Mirna Therapeutics, Inc. Procédés et compositions impliquant des miarn dans des cellules souches cancéreuses
WO2010061396A1 (fr) * 2008-11-30 2010-06-03 Rosetta Genomics Ltd. Procédés pour détecter une susceptibilité augmentée au cancer
WO2012005572A1 (fr) * 2010-07-06 2012-01-12 Interna Technologies Bv Miarn et ses utilisations diagnostiques et thérapeutiques pour des maladies ou des états associés au mélanome, ou pour des maladies ou des états associés à la voie braf activée
US20140088170A1 (en) 2012-08-29 2014-03-27 City Of Hope Differentially expressed microrna molecules for the treatment and diagnosis of cancer

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
BAO, BO-YING ET AL.: "Polymorphisms inside microRNAs and microRNA target sites predict clinical outcomes in prostate cancer patients receiving androgen-deprivation therapy.", CLINICAL CANCER RESEARCH, vol. 17, no. 4, 2011, pages 928 - 936., XP055097252, Retrieved from the Internet <URL:https://www.researchgate.net/ publication/49675441_Polymorphisms_inside_MicroRNAs_and_MicroRNA_Target_Sites_Pr edict_Clinical_Outcomes_in_Prostate_Cancer_Patients_Receiving_Androgen- Deprivation_Therapy>> *
FENG ET AL., ONCOTARGET, vol. 5, no. 14, 2014
GRIFFITHS-JONES ET AL., NUCL ACIDS RES, vol. 36, 2008, pages D154 - D158
HU ET AL., PLOS ONE, vol. 10, no. 5, 14 May 2015 (2015-05-14), pages e0121175
HUANG ET AL., INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, vol. 4, no. 6, 3 December 2014 (2014-12-03), pages 1599 - 605
LI ET AL., BREAST CANCER RESEARCH, vol. 16, 2014, pages 473
OFEK P. ET AL.: "In vivo delivery of small interfering RNA to tumors and their vasculature by novel dendritic nanocarriers", FASEB J, vol. 24, 2010, pages 3122 - 34, XP055085077, DOI: 10.1096/fj.09-149641
PEER, D. ET AL.: "Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target", SCIENCE, vol. 319, 2008, pages 5863
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SARFATI G. ET AL.: "Targeting of polymeric nanoparticles to lung metastases by surface-attachment of YIGSR peptide from laminin", BIOMATERIALS, vol. 32, no. 1, 2011, pages 152 - 61, XP027493699, DOI: 10.1016/j.biomaterials.2010.09.014
See also references of EP3322423A4
YU ET AL., CANCER RESEARCH, vol. 70, no. 14, 15 July 2010 (2010-07-15), pages 6015 - 25

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017223492A1 (fr) * 2016-06-23 2017-12-28 Massachusetts Institute Of Technology Compositions, dispositifs, et procédés d'administration d'arnmicro
CN107881239A (zh) * 2017-12-27 2018-04-06 广西壮族自治区肿瘤防治研究所 血浆中与结直肠癌转移相关的miRNA标志物及其应用
CN108004323A (zh) * 2017-12-27 2018-05-08 广西壮族自治区肿瘤防治研究所 组织中与结直肠癌转移相关的miRNA标志物及其应用
CN108004323B (zh) * 2017-12-27 2021-03-30 广西壮族自治区肿瘤防治研究所 组织中与结直肠癌转移相关的miRNA标志物及其应用
CN107881239B (zh) * 2017-12-27 2021-04-13 广西壮族自治区肿瘤防治研究所 血浆中与结直肠癌转移相关的miRNA标志物及其应用
WO2020002565A1 (fr) 2018-06-27 2020-01-02 Sabine Bauer Implants pour recruter et retirer des cellules tumorales circulantes
WO2021122803A2 (fr) 2019-12-17 2021-06-24 Cirlo Gmbh Ensembles dispositifs de cathéter allongés de forme tubulaire destinés à entrer en interaction avec des composants de fluides corporels, procédé de récupération de cellules, d'agrégats de cellules et d'exosomes à partir d'un dispositif de cathéter allongé de forme tubulaire et ensembles dispositifs de cathéter allongés de forme tubulaire intelligents pour surveiller l'interaction avec des composants de fluides corporels

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